Diagnosis of infection in the lungs of patients

ABSTRACT

The present invention relates to methods for detecting P aeruginosa infection and bacterial burden in the lungs of patients who are at risk for P. aeruginosa infections, especially including patients with Cystic Fibrosis (CF). The present method provides numerous tests (breath, blood, urine) which are readily administered to a patient that will sensitively and specifically detect the presence and extent of lung infection P. aeruginosa (both mucoid and non-mucoid), and allow monitoring of bacterial load as a parameter in monitoring treatment.

This application is a continuation of PCT/US08/05412 dated Apr. 25,2008, which claims benefit to 60/958,615 dated Jul. 6, 2007, whichclaims benefit to 60/927,670 dated May 4, 2007, which claims benefit to60/926,191 dated Apr. 25, 2007.

The present application claims priority from U.S. provisionalapplications Ser. No. 60/926,191, filed Apr. 25, 2007, Ser. No.60/927,670, filed May 4, 2007 and Ser. No. 60/958,615, filed Jul. 6,2007, each of which applications is incorporated by reference herein inits entirety.

RELATED APPLICATIONS AND FEDERALLY SPONSORED RESEARCH

The present invention was made with government support under Grant Nos.A1 050825 and AI 31139 awarded by the National Institutes of Health.Consequently, the Government retains rights in this invention.Thisinvention was made with government support under grant numbers A1 050825and A1 31139, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods for detecting P aeruginosainfection and bacterial burden in the lungs of patients who are at riskfor P. aeruginosa infections, especially including patients with CysticFibrosis (CF). The present method provides numerous tests (breath,blood, urine) which are readily administered to a patient that willsensitively and specifically detect the presence and extent of lunginfection P. aeruginosa (both mucoid and non-mucoid), and allowmonitoring of bacterial load as a parameter in monitoring treatment.

In addition, the present invention also relates to a method fordetecting non-mucoid and mucoid P. aeruginosa infection and its burdenin the lungs of patients with Cystic Fibrosis (CF), specifically to amethod which is a special breath test that will sensitively andspecifically detect the presence and extent of lung infection bynon-mucoid and mucoid P. aeruginosa, and allow monitoring of mucoidbacterial load as a parameter in treatment. It is achieved by sensitivedetection of volatile (or non-volatile) products of the denitrificationpathway that occurs in P. aeruginosa. These can be detected either asisotopically labeled products from labeled precursors (such as ¹⁵NO₃ or¹⁵NO₂).

BACKGROUND OF THE INVENTION

Cystic fibrosis is a major genetic disease in many populations, and hasbeen transformed from a life ending, to a life-shortening diseasethrough aggressive therapy especially that aimed at the major pathogenin CF P. aeruginosa. However, there is no currently available method torapidly determine whether a CF patient is actively infected with P.aeruginosa or to assess whole-lung bacterial levels.

The progressive lung damage and deterioration of respiratory function incystic fibrosis (CF) arises from a characteristic pattern of bacterialcolonization of the lung, with chronic Pseudomonas aeruginosa infectionsbeing centrally important to the lung pathology and progressive tissuedamage in CF patients. More than 80% of CF patients over 26 years oldare chronically infected with P. aeruginosa, and these infectionspersist, despite aggressive antibiotic therapy. After initial infectionwith wild-type, non-mucoid strains, exemplified by strains such as PAO1,conversion of P. aeruginosa to the mucoid phenotype in the CF hostoccurs and increases bacterial resistance not only to host clearance anddefense mechanisms, but also to antibiotic interventions. The mucoidphenotype is characterized by production of large amounts of theexopolysaccharide alginate. Mucoid conversion is concomitant with theestablishment of chronic bacterial colonization and increasesinflammation and lung damage, while causing pulmonary function todecline. Furthermore, alginate modulates host defenses to allow apersistent infection. Accordingly, mucoid conversion results in a poorprognosis for CF patients to heal

Rapidly knowing if mucoid conversion has occurred, and the extent ofmucoid pseudomonas burden would greatly improve therapies. However,there is no currently available method to rapidly determine whether a CFpatient is infected, or colonized with mucoid R aeruginosa or to assesswhole-lung mucoid bacterial levels. Currently, culture of sputumsamples, or bronchioalveolar lavage, together with phenotypic (andgenotypic) analysis. We propose a novel detection system that willrevolutionize mucoid bacterial detection, and so management of CF. Bymaking key modifications to current H pylori breath test, we willfurnish a specific and selective test for the presence and bacterialload of mucoid P aeruginosa in CF lungs.

An H. pylori breath test is presently available for detecting H. pylorilevels in the gut of patient. The basis for the H pylori breath test isthat a drink containing labeled urea (carbon-13 or carbon-14) isingested, with the urea rapidly encountering urease-positive H. pyloribacteria in the gut (if the bacteria is present) that break down theurea into labeled CO₂ that is detected in the breath about 15 minutesafterwards.

P. aeruginosa also expresses urease¹⁻⁴, has the urease urea-ureG genecluster whose expression is modifiable (Deretic & Timmins, unpublishedmicroarray data), and consequently, is susceptible to detection bybreath tests if the appropriate technology is developed and applied.Because P. aeruginosa is so exquisitely associated with the CF lung, andnot the normal population, by targeting the CF patient, high specificitycan be achieved.

SUMMARY

The present invention provides a method for detecting mucoid andnon-mucoid P. aeruginosa in a patient with cystic fibrosis (CF), whereinan isotopically labeled compound such as a nitrate salt, a nitrite salt,nitric oxide (NO) donor, glycine and/or urea is delivered to thepatient, either orally, via an intravenous route or preferably, directlyto the lungs of the CF patient in an inhaled dosage form. In thepresence of P. aeruginosa, the labeled compounds are converted tolabeled nitrate, nitrate, nitric oxide, nitrogen, nitrous oxide,cyanate, thiocyanate, hydrogen cyanide (HCN), carbon dioxide (CO₂) whichis detected upon exhalation or in the blood, serum, plasma or urine ofthe patient or subject. The amount of conversion can be used as an assayof overall bacterial load, response to treatments, and other importantparameters to the management of CF, including the extent to whichnon-mucoid bacteria have been converted to the more potent and dangerousmucoid bacteria.

The present invention relates to a method for identifying the existenceof a P. aeruginosa infection in the lungs of a subject (CF patient) tobe diagnosed and whether the infection has advanced to a moreproblematic mucoid stage from a non-mucoid stage.

According to the present invention there is provided a method for thediagnosis of Psedomonas aeruginosa in the lungs of a CF patient orsubject, including the steps of:

(i) administering at least one isotopically labeled compound selectedfrom the group consisting of a pharmaceutically acceptable nitrate salt,a pharmaceutically acceptable nitrite salt, NO gas, a nitric oxide (NO)donor/precursor such as a pharmaceutically acceptable S-nitrosothiolcompound (e.g., an alkylthionitrile, S-glutathione,S-nitroso-N-acetyl-penicillamine), urea and/or glycine (isotopicallylabeled metabolic precursors referred to in context collectively as“active agents”, “active compounds” “isotopically labeled active agentsor compounds”) to said patient such that, if the patient has a P.aeruginosa infection in the lungs of the subject, the said isotopicallylabeled compound is metabolized to at least one isotopically labeledmetabolite (preferably, a gas) selected from the group consisting ofnitrate, cyanate, thiocyanate (for detection in urine), or N₂, NO, N₂O,CO₂ and HCN (for detection in the breath of the subject) and

(ii) the metabolite produced in step (i) is quantified, such that theamount of isotopically labeled metabolite is evidence of a P. aeruginosainfection. A positive reading of any of the isotopically labeledmetabolites may indicate the presence of a P. aeruginosa infection inthe lungs of the subject and a high positive reading of HCN, cyanate orthiocyanate relative to other metabolites identified and quantified willsupport the likelihood that the P. aeruginosa infection containssignificant mucoid P. aeruginosa.

In the present invention the metabolites are measured in the urine ofthe subject or by analyzing a plurality of exhaled breaths of thesubject to determine a concentration of said metabolic/cleavageproduct(s), the concentration indicating the presence or absence of P.aeruginosa and high levels of glycine metabolites (cyanate, thiocyanatein urine or HCN in breath) compared to a control, evidencing thepresence of mucoid P. aeruginosa. Optional steps include fitting theconcentrations to a curve; and analyzing the curve or plateau todetermine the extent of infection.

Preferably, the metabolite obtained above is determined by analyzingexhaled breath of the subject. Analyzing the exhaled breath of thesubject is repeated substantially at a particular time or times until apredetermined time period (which can range from as little as severalbreaths in a short period to a number of breaths in several minutes ormore) has elapsed. This can be done by collecting breaths from a subjectover a predetermined period of time or times. The predetermined periodof time may be determined by analyzing the activity of control subjectswith P. aeruginosa infections to cleave/metabolize isotopically labelednitrate, nitrite, S-nitrosothiol compound, urea and/or glycine measuringthe concentration of isotopically labeled nitrogen, nitric oxide (NO),nitrous oxide (N₂O), carbon dioxide and/or hydrogen cyanide which isfound in the exhaled breath of the control subjects as a function oftime after administration of the metabolic precursor.

The urine or exhaled breath of the subject is analyzed for content ofthe isotopically labeled element which evidences the residence of P.aeruginosa. In certain embodiments related to a breath analysis, thestep of analyzing the exhaled breath of the subject is repeatedsubstantially until a particular accuracy for analyzing the data isreached. In certain preferred aspects of the invention, a ratio of theconcentration of an isotopically labeled element to a non-isotopicallylabeled element measured for a sample taken at one or more predeterminedtimes and for one or more predetermined periods is calculated and agraph of the ratio (y-axis) as a function of time (x-axis) is provided.Preferably, the breath of the subject is analyzed by an infrared laserspectrometer or a mass spectrometer to determine concentrations ofisotopically labeled elements and non-isotopically labeled elements. Ina particularly preferred clinical diagnostic embodiment, a breath testaccording to the present invention will be designed to determine theexistence of P. aeruginosa and in particular, mucoid P. aeruginosa, inthe lungs of a CF patient or subject.

According to further preferred embodiments of the present invention, thenitrate, nitrite, NO gas, or S-nitrosothiol (stable NO precursorcompound) urea and glycine are all isotopically-labelled. In the case ofisotopically labeled nitrate, nitrite or the S-nitrosothiol compound,each of these compounds is labeled with an isotopically labeled nitrogen(nitrogen-15) or oxygen (oxygen-17, oxygen-18), preferably labeledNitrogen, and if P. aeruginosa is present in the lungs of a subject, thecleavage/metabolic product is an isotopically labeled (nitrogen-15) gassuch as N₂, or an isotopically labeled (nitrogen-15) or (oxygen-17,oxygen-18) gas in the case of N₂O or NO (note that a nitrogen-15 labeledgas is preferred), which may be measured/analyzed in the breath of thesubject. In the case of urea, this may be isotopically labeled withnitrogen-15, carbon-13, oxygen-17 or oxygen-18, preferably withnitrogen-15 and/or carbon-13, such that if P. aeruginosa is present inthe lungs of the subjected to be tested, the urea will becleaved/metabolized to isotopically labeled ammonia and/or carbondioxide. Using isotopically labeled urea (labeled accordingly), theresulting ammonia is nitrogen-15 labeled, whereas the carbon dioxide iscarbon-13 labeled and/or oxygen-17 and/or oxygen-18 labeled. In the caseof glycine, the glycine is labeled with nitrogen-15 or carbon-13 ormixtures thereof, such that the resulting HCN gas, cyanate orthiocyanate (urine analysis) is isotopically labeled with nitrogen-15,carbon-13 or mixtures thereof. It is noted that although it ispreferable to analyze for isotopically labeled (gaseous) nitricoxide/nitrous oxide/Nitrogen oxide/Nitrogen (nitrate, nitrite, NOprecursor cleavage products/metabolites) and/or carbon dioxide (ureacleavage products/metabolites) and/or hydrogen cyanide (HCN) in thebreath of a subject, it is also possible to analyze for isotopicallylabeled nitrite, nitrate, cyanate or thiocyanate or even dissolvedCO₂/bicarbonate in the blood (plasma, serum) or urine of a subject asalternative cleavage products.

It is noted that in the case of the use of isotopically labeled urea,urea may be labeled with isotopically labeled carbon-13, nitrogen-15,oxygen-17 and/or oxygen 18, wherein the cleavage products to be analyzedfrom urea being acted upon by P. aeruginosa urease are isotopicallylabeled carbon dioxide and/or ammonia. In the case of isotopicallylabeled glycine, the glycine may be labeled with isotopically labeledcarbon-13, nitrogen-15 or mixtures thereof wherein the cleavage productsto be analyzed from glycine being acted upon by the cyanide synthasepathway in P. aeruginosa to form hydrogen cyanide gas (to be analyzed bybreath analysis) and/or cyanate and/or thiocyanate anion (to be analyzedby blood/serum/plasma or urinalysis).

The present invention also relates to pharmaceutical compositionscomprising effective amounts of nitrate salts (sodium or potassium),nitrite (sodium or potassium), S-nitrosothiol compound (e.g., analkylthionitrile, S-glutathione, S-nitroso-N-acetylpenicillamine), ureaand/or glycine in pulmonary dosage form. A preferred compound comprisesan effective amount of glycine or a pharmaceutically acceptable saltthereof, preferably glycine and urea or pharmaceutically salts thereof,and more preferably a nitrate salt, a nitrite salt and/or anS-nitrosothiol compound in combination with the glycine and urea (orpharmaceutical salts thereof). This composition comprises thesecomponents in effective amounts, in combination with a pharmaceuticallyacceptable carrier, additive or excipient, as well as a propellant andoptionally, a solvent and/or a dispersant. The composition, in its mostpreferred form, is adapted for pulmonary administration to the lungs ofa subject or patient.

The present invention also relates to a composition in oral dosage formcomprising effective amounts of at least one compound selected from thegroup consisting of a nitrate salt, nitrite salt, an NO precursorcompounds such as an S-nitrosothiol compound (e.g., an alkylthionitrile,S-glutathione, S-nitroso-N-acetylpenicillamine) and mixtures thereof, incombination with an effective amount of urea and glycine (or apharmaceutically acceptable salt thereof), all isotopically labeled,optionally in combination with a pharmaceutically acceptable carrier,additive or excipient and further optionally in combination with aneffective amount of a urease inhibitor. The urease inhibitor ispreferably included in oral dosage forms in order to inhibit the ureaseof H. pylori which may reside in the stomach/gastrointestinal tract of asubject and which can reduce and/or negate the effects of urea inhelping to diagnose a P. aeruginosa infection in the lungs of a patient.

In another aspect of the invention, a kit for diagnosing P. aeruginosain a subject or patient comprises a composition comprising at least onecompound selected from the group consisting of a nitrate salt, nitritesalt, a NO precursor compound such as a S-nitrosothiol compound (e.g.,an alkylthionitrile, S-glutathione, S-nitroso-N-acetylpenicillamine) andmixtures thereof, in combination with an effective amount of urea andglycine (or a pharmaceutically acceptable salt thereof), allisotopically labeled, optionally in combination with a pharmaceuticallyacceptable carrier, additive or excipient and further optionally incombination with an effective amount of a urease inhibitor and mixturesthereof in oral or pulmonary dosage form, a collection bag or vial tocollect exhaled breaths from said subject or patient; and an optionalinstruction manual. In preferred aspects, the composition will comprisea combination of at least one compound selected from the groupconsisting of a nitrate salt, nitrite salt, an S-nitrosothiol compound(e.g., an alkylthionitrile, S-glutathione,S-nitroso-N-acetylpenicillamine) in combination with an effective amountof urea and glycine (or a pharmaceutically acceptable salt thereof), allisotopically labeled in pulmonary dosage form. In oral dosage form, sucha combination of agents is combined with an optional effective amount ofa urease inhibitor as otherwise disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram representing how isotopically labeled compoundsmay be delivered to the lungs of a patient.

FIG. 2 shows a diagram representing how P. aeruginosa converts labeledglycine or urea to labeled HCN in the lung. Detection of labeled HCNindicates mucoid P. aeruginosa levels.

FIG. 3. shows a diagram representing how mucoid P. aeruginosa convertslabeled glycine to labeled cyanate. Detection of labeled cyanate, or itsmetabolites such as thiocyanate, in the blood or urine at significantlevels indicate the existence of significant levels of mucoid P.aeruginosa.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used to describe the present invention. Ininstances where a term is not defined specifically, that term shall beaccorded the ordinary meaning ascribed to the term by those of ordinaryskill in the art when used within context.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential.

The methods and processes illustratively described herein suitably maybe practiced in differing orders of steps, and that they are notnecessarily restricted to the orders of steps indicated herein or in theclaims. As used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, a reference to “an isotopicallylabeled element” includes a plurality (for example, two or moreelements) of such elements, and so forth. Under no circumstances may thepatent be interpreted to be limited by any statement made by anyExaminer or any other official or employee of the Patent and TrademarkOffice unless such statement is specifically and without qualificationor reservation expressly adopted in a responsive writing by Applicants.

The term “patient” or “subject” is used to describe an individualsubject or patient, a mammal, generally a human, who is at risk fordeveloping a P. aeruginosa infection, in particular patients with cysticfibrosis. In the present invention, the term patient or subjectgenerally refers to a patient or subject who has cystic fibrosis or isat least suspected of having cystic fibrosis.

The term “active agents”, “active compounds” “isotopically labeledactive agents or compounds” describes, within context, isotopicallylabeled metabolic precursors which are acted on by enzyme pathways of P.aeruginosa to produce identifiable and distinguishable metabolites whichcan be analyzed using conventionally available analytic tools andmethodologies. Active agents according to the present invention includepharmaceutically acceptable nitrate salts (sodium and/or potassium),pharmaceutically acceptable nitrite salts (sodium and/or potassium), NOgas, a nitric oxide (NO) donor/precursor compound such as apharmaceutically acceptable S-nitrosothiol compound (e.g., analkylthionitrile, S-glutathione, S-nitroso-N-acetylpenicillamine), ureaand/or glycine or pharmaceutically acceptable salts thereof. In thepresent invention, if a CF patient has a P. aeruginosa infection in hisor her lungs, the isotopically labeled compound, which is delivered tothe lungs and is metabolized to at least one isotopically labeledmetabolite (preferably, a gas) selected from the group consisting ofnitrate, nitrite, dissolved NO, cyanate, thiocyanate,bicarbonate/carbonate (for detection in blood, serum, plasma or urine),or N₂, NO, N₂O, CO₂ and HCN (for detection in the breath of thesubject).

The terms “cleavage products” and “metabolites” are used in thespecification synonymously to refer to isotopically labeled compoundsthat are formed by the action of P. aeruginosa on isotopically labeledactive agents or active compounds as otherwise described herein.

The term “effective” refers to an amount of an active agent or compound(i.e., isotopically labeled nitrate, nitrite, NO gas, NO precursorcompound (an S-nitrosothiol compound, e.g., an alkylthionitrile,S-glutathione, S-nitroso-N-acetyl-penicillamine), urea and/or glycine)which is sufficient to produce a detectable level of a cleavage productor products, without an untoward level of adverse side effects, such astoxicity, irritation, allergy or hypersensitivity responses in apatient. The level of any such side effects should be commensurate withacceptable risk/benefit ratios. In the present invention, the termeffective is used to describe an amount of a substrate or othersubstance or component which is used to effect an intended result withinthe context of its use.

The term “cystic fibrosis” or “CF” is used to describe one of the mostcommon grave genetic (inherited) diseases. CF affects the exocrineglands and is characterized by the production of abnormal secretions,leading to mucous build-up. It is also characterized by severerespiratory problems including chronic infections of the lungs (P.aeruginosa), emphysema and progressive respiratory insufficiency.Chronic P. aeruginosa infection often results in accumulation of mucusin the lungs as the infection progresses from a non-mucoid to a mucoidphase. P. aeruginosa also expresses the enzyme cyanide synthase, fromthe cyanide synthase gene cluster (hcnA-C) that oxidizes glycine tohydrogen cyanide, and the expression of this enzyme is vastlyupregulated in mucoid as opposed to non-mucoid P. aeruginosa. Inaddition to causing damage in the lungs, the accumulation of mucus canimpair the pancreas and, secondarily, the intestine. Mucous build-up inlungs tends progressively to impair respiration. Without treatment, CFresults in death for 95% of affected children before age 5.

With diligent medical care, patients with CF are surviving even beyondmiddle age. Early diagnosis of CF is of great importance. Early andcontinuing treatment of CF is essential for long-term survival. However,as more people with CF survive childhood, new problems are emerging,including urine leakage, which occurs most severely when their chestdisease is most severe.

CF is inherited in an autosomal recessive manner and affects both boysand girls. One in 400 caucasian couples is at risk for having childrenwith CF and their risk with each pregnancy is 1 in 4, with an overallrisk that their child will have CF is 1 in 1600. The treatment of CFincludes physical therapy to loosen the mucus in the lungs, pancreaticenzymes, and medications to fight dangerous infections of the lungs. CFis caused by mutation in the gene encoding CFTR (cystic fibrosisconductance regulator) on chromosome 7. The most common mutation,DeltaF508, results in the production of a misfolded CFTR protein that isretained in the endoplasmic reticulum and targeted for degradation.

The term “Pseudomonas aeruginosa” or P. aeruginosa is used to describe ag, aerobic, rod-shaped bacterium with unipolar motility, which commonlyresides in the lungs (and other tissue) of patients with cysticfibrosis. An opportunistic human pathogen, P. aeruginosa is also anopportunistic pathogen of plants. P. aeruginosa is the type species ofthe genus Pseudomonas.

P. aeruginosa secretes a variety of pigments, including pyocyanin(blue-green), fluorescein (yellow-green and fluorescent, now also knownas pyoverdin), and pyorubin (red-brown). P. aeruginosa is oftenpreliminarily identified by its pearlescent appearance and grape-likeodor in vitro. In the past, definitive clinical identification of P.aeruginosa often includes identifying the production of both pyocyaninand fluorescein as well as its ability to grow at 42° C. Althoughclassified as an aerobic organism, P. aeruginosa is considered by manyas a facultative anaerobe as it is well adapted to proliferate inconditions of partial or total oxygen depletion. This organism canachieve anaerobic growth with nitrate as a terminal electron acceptor,and in its absence it is also able to ferment arginine bysubstrate-level phosphorylation. Adaptation to microaerobic or anaerobicenvironments is essential for certain lifestyles of P. aeruginosa, likeduring lung infection in cystic fibrosis patients where thick layers ofalginate surrounding bacterial mucoid cells can limit the diffusion ofoxygen.

The term “coadministration” is used to describe the administration of atleast two active (isotopically labeled) compounds, in this case at leasturea and glycine, and preferably urea and glycine in combination with atleast one further compound selected from a nitrate salt, a nitrite salt,NO gas or a NO stable compound such as an S-nitrosothiol compound (e.g.,an alkylthionitrile, S-glutathione, S-nitroso-N-acetyl-penicillamine),all in effective amounts. Although the term coadministration preferablyincludes the administration of at least two active compounds to thepatient at the same time, it is not necessary that the compoundsactually be administered at the exact same time, only that amounts ofcompound will be administered to a patient or subject such thateffective concentrations are found in the blood, serum or plasma, or inthe pulmonary tissue at the same time.

The term “cleaves”, “cleavage product” or “metabolite” is used withreference to the fact that an enzyme of P. aeruginoa can break down ormetabolize at least one chemical bond of the active isotopically labeledcompounds, forming “cleavage products” (e.g, nitrate, nitrite, nitricoxide, nitrous oxide, nitrogen gas, carbon dioxide, ammonia, hydrogencyanide, cyanate or thiocyanate, etc), by chemical processes including,but not limited to, hydrolysis, oxidation/reduction and/ordenitrification. In the present invention, the concentration of thecleavage product or products (in the breath, whole blood, serum, plasmaor urine of a subject or patient) indicates the level of activity of P.aeruginosa species in the lungs (or other tissues) of the subject, whichcan be used to determine a diagnosis of infection by P. aeruginosa, andthe intensity of the infection. A positive diagnosis indicates that P.aeruginosa is present in the lungs (or other tissues) of the subject andcan indicate whether a P. aeruginosa infection is primarily a mucoidinfection, which is a particularly dangerous infection for CF patients.In preferred aspects of the invention, the method for diagnosis ispreferably a “breath test”, due to its ease of use by analyzing thebreath of the subject for evidence of cleavage product.

Examples of appropriate labels for the substrate, and hence for thecleavage product or products, are those which can be detected by anappropriate measuring instrument, conventional spectroscopic includingUV and IR, mass spectroscopic, radiochemical techniques, electrochemicalanalysis (nitrates and nitrites in serum, blood and urine), laserspectroscopy, GC/mass spectroscopy, but which are substantially notharmful or toxic to the subject including, but not limited to, carbon-13or carbon-14, oxygen-17, oxygen-18 or nitrogen-15, isotope-labeling. Anisotope is a form of an element, such as carbon, with a specific mass.For example, carbon-12 has a mass of 12 atomic mass units. The term“isotope-labeling” means that the naturally more abundant isotope ofeach of these elements is at least partially replaced by a less abundantisotope. For example, the naturally more abundant carbon-12 atoms couldbe at least partially replaced by the less abundant carbon-13 atoms,permitting the cleavage product or products which carry the label to bemore easily detected, since the less abundant isotope can bedistinguished from the naturally more abundant isotope. Furthermore, theadvantage of certain isotopes such as carbon-13 is that they are stable,so that they are not radioactive, unlike isotopes such as carbon-14.Note that carbon 14 may be used in rare instances in an animalexperimentation setting, but is inappropriate for use in a therapeuticsetting. Therefore, preferably stable, non-radioactive isotopes such ascarbon-13 are used as labels in the present invention.

The term “isotopically labeled” shall mean isotopically labeled withcarbon-13, nitrogen-15, oxygen-17 or oxygen-18 at positions on the whichgive rise to isotopically labeled cleavage products as otherwisedisclosed herein after exposure to P. aeruginosa denitrification pathway(nitrates, nitrites, nitric oxide precursors), urease (urea), andcyanide synthase (glycine), respectively.

The term “radiometric” or “radioactive ratio” is used to describe aratio of isotopically labeled hydrogen cyanide, cyanate or thiocyanatewhich is analyzed in a CF patient having a P. aeruginosa infection whichis principally a mucoid producing infection compared to an infection ina CF patient which is principally a non-mucoid producing infection.Because mucoid P. aeruginosa expresses cyanide synthase to a far greaterextent than does non-mucoid P. aeruginosa, this difference may beutilized to establish that a P. aeruginosa infection is a mucoidproducing infection. By comparing concentrations of isotopically labeledHCN in the breath of CF patients tested using the methods of presentinvention, or cyanate or isocyanate levels in the blood, serum or urineof the tested CF patients with controls established for these samecleavage products for patients 1) without P. aeruginosa infection or 2)with a non-mucoid P. aeruginosa infection, one may readily assess thelikelihood that the test patient has a mucoid P. aeruginosa infectionbased upon the results obtained. In the present invention, a radioactiveratio of the test patient of at least about 1.25 times, at least about1.3, at least about 1.4, at least about 1.5, at least about 1.65, atleast about 1.75, at least about 1.85, at least about 2.0, at leastabout 2.1, at least about 2.25, at least about 2.5, at least about 3.0,at least about 5.0 evidences the existence of a more severe mucoid P.aeruginosa infection in the lungs of the CF patient tested.

According to the present invention there is provided a method for thediagnosis of Psedomonas aeruginosa in the lungs of a CF patient orsubject, including the steps of:

(a) administering at least one isotopically labeled compound selectedfrom the group consisting of a pharmaceutically acceptable nitrate salt(e.g., sodium, potassium), a pharmaceutically acceptable nitrite salt(sodium potassium), NO gas, a NO (nitric oxide) precursor such as anS-nitrosothiol (e.g., an alkylthionitrile, S-glutathione,S-nitroso-N-acetyl-penicillamine), urea and/or glycine to said patientsuch that, if the patient has a P. aeruginosa infection in the lungs ofthe subject, the said isotopically labeled compound is metabolized to atleast one isotopically labeled cleavage product selected from the groupconsisting of nitrate, nitrite, N₂, NO, N₂O, CO₂, HCN, cyanate orthiocyanate and quantified. A positive reading of any of the cleavageproducts may indicate the presence of a P. aeruginosa infection and ahigh positive reading of HCN, cyanate or thiocyanate compared to acontrol value will support the likelihood of a mucoid P. aeruginosainfection in the patient's lungs. Once an infection is established,appropriate therapy (antibiotic therapy) and/or remediation may begin.

In preferred aspects of the invention, the isotopically labeled compoundis a mixture of an effective amount of urea and glycine, and optionallyat least one additional isotopically labeled compound selected from thegroup consisting of a nitrate salt, nitrite salt and a NO precursor inpulmonary dosage form for administration to the lungs of the CF patientto be tested and the cleavage products are gases (e.g., NO, N₂O, N₂,CO/CO₂, HCN) which are measured/analyzed for in the breath (a pluralityof breaths) of the patient, the concentration of isotopically labeledgas indicating the presence or absence of a P. aeruginosa bacterialinfection and the concentration of HCN indicating the presence orabsence of a significant mucoid P. aeruginosa infection in the lungs ofthe CF patient tested. Optional steps include fitting the concentrationsto a curve; and analyzing the curve or plateau to determine the extentof infection.

The present invention is a diagnostic test, preferably a breath test,which can be used to rapidly and accurately detect the presence of P.aeruginosa, and in certain preferred aspects, mucoid P. aeruginosa inthe lungs of a CF patient or subject. Specifically, the presentinvention can be used to diagnose the presence of P. aeruginosa by theadministration (preferably pulmonary/intratracheal administration) of asafe and effective amount of isotopically labeled nitrate salt (e.g.,sodium, potassium), nitrite salt (sodium potassium), a NO (nitric oxide)precursor such as an S-nitrosothiol (e.g., an alkylthionitrile,S-glutathione, S-nitroso-N-acetylpenicillamine), urea and/or glycine toa subject and then detecting the concentration of at least one of thecleavage/metabolic products of these compounds preferably as gases inbreath exhaled by the subject.

In preferred aspects of the invention, several breaths taken at apredetermined time after administration of one or more of these products(preferably urea and glycine and optionally, at least one compoundselected from a nitrate salt, a nitrite salt or a NO precursor compound)is sufficient for providing a measure of isotopically labeled Nitrogen,carbon or oxygen. If the ratio of measured isotopically labeled elementto non-isotopically labeled element is greater than a predeterminedvalue, the existence and in many cases the severity of the P. aeruginosainfection may be readily determined. In addition, if a concentration ofHCN is determined to be greater than a predetermined value or ratio, theexistence of a mucoid P. aeruginosa infection may be readily determined.Analysis may result in therapeutic intervention on the patient beingtaken.

Thus, in the case of nitrate salts, nitrite salts or NO precursorcompounds detection of isotopically labeled nitrogen-15 or oxygen-17/18in nitric oxide, N₂O or N₂ gas in the exhaled breath of the subjectafter a suitable period of time after administration (depending upon theroute of administration) can provide diagnostic information about theexistence of P. aeruginosa in the subject's lungs (or other tissues). Inthe case of urea, isotopically labeled carbon-13, nitrogen 15 or oxygen17/18 in carbon dioxide or ammonia, or both cleavage products, measuredin the exhaled breath of the patient or subject, after a suitable timeperiod has elapsed, can also provide diagnostic information about theexistence of P. aeruginosa infection in the subject's lungs (or othertissues). In the case of glycine, isotopically labeled carbon-13 ornitrogen-15 in HCN measured in the exhaled breath of the patient orsubject, after a suitable time period has elapsed, can providediagnostic information about the existence of a mucoid P. aeruginosainfection in the lungs of the patient or subject. In each instance, theisotopically labeled cleavage products are compared with a baselinevalue obtained for the patient prior to administration of the activecompounds.

In a preferred embodiment, isotopically labeled urea and glycine andoptionally, at least one additional compound selected from the groupconsisting of a nitrate salt, a nitrite salt and a NO precursor compoundare administered to the subject to be tested for P. aeruginosa infectionand, depending on the existence of cleavage products or their isotopicelements (or their absence) measured in the exhaled breath of thesubject to be tested, diagnostic information may be used to determinewhether or not there is a P. aeruginosa infection in the lungs (or othertissues) of the subject tested and whether, if there such an infection,whether that infection is a mucoid P. aeruginosa infection. In preferredaspects of the invention, a single breath or several breaths taken at apredetermined time after administration of the isotopically labeledcompounds may be used to determine the existence or absence of an activeP. aeruginosa infection and optionally, whether or not it is a mucoidinfection.

In an alternative aspect of the invention, in order to determine that aP. aeruginosa infection is present in the lungs (or other tissues) ofthe tested patient, measurements of isotopically labeled cleavageproducts in the breath of the tested subject are made and theconcentration of isotopically labeled elements is compared withconcentrations of the naturally occurring nitrogen-14, carbon-12 and/oroxygen-16 atoms in the breath of the tested subject. Thus, in thisaspect of the invention, a control or baseline may be established forthe patient or subject to be tested. A single breath or a number ofbreaths taken at a predetermined time based upon the time ofadministration of the isotopically active compounds to the subject maybe used to diagnose the subject. Alternatively, a number of breaths atdifferent times may be used in diagnosis. The ratio of an isotopicallylabeled element(s) to non-isotopically labeled element(s) may bedetermined and compared to a predetermined reference or control value(ratio of the same elements) determined from the subject prior toadministration of the isotopically labeled compounds. A singlemeasurement obtained from the subject which evidences a ratio above thereference ratio may be evidence of the existence of infection and/ormucoid phenotype. A measurement of approximately the reference ratiowill be evidence of that an infection does not exist.

In a preferred embodiment, a combination of an isotopically labelednitrate, nitrite or NO precursor (labeled with for example, nitrogen 15,oxygen 17 or 18) may be used in combination with an isotopically labeledurea (C-13) and an isotopically labeled glycine (N-15) in pulmonarydosage form. In this aspect of the invention, the active compounds aredelivered to the lungs of the CF patient or subject, and the patient'sexhaled breaths are collected and analyzed to determine the existence ofisotopically labeled elements at levels above baseline (control), suchthat the metabolism of the active compounds is detected (elevatedisotopic elements) and the existence of a P. aeruginosa infection isconfirmed. The existence of elevated levels of HCN gas and/or isotopicelement from the administered glycine will evidence, along with theother cleavage products that the P. aeruginosa is a mucoid infection.

In alternative embodiments, a number of breaths at different times maybe taken from the subject and a graph or curve may be generated showingthe ratio of the isotopically labeled element to the naturally occurringelements in the breath of the tested subject as a function of time. Acurve showing an increase in the ratio of the isotopically labeledelement to non-isotopically labeled element over time (compared to acontrol with no infection) is evidence of the existence of a P.aeruginosa infection. The concentrations of isotopically labeledelements in the samples are compared to a standard ratio which may beobtained from a control group, or more preferably, from the subjectprior to administration of the active compounds to the subject pursuantto the diagnostic test.

A curve may be fitted to the measured concentrations and then analyzed,preferably by determining the rate of rise of the curve, or by themagnitude of the plateau. Such an analysis indicates the level ofactivity of P. aeruginosa species in the subject, which can be used todiagnose the presence of P. aeruginosa in the lungs (or other tissues)of the subject and the phenotype (non-mucoid or mucoid) of the P.aeruginosa infection.

Preferably at least a majority of the exhaled breaths, and mostpreferably every exhaled breath at a predetermined time for apredetermined period, is sampled for that period or until thedetermination of the level of P. aeruginosa activity, if present, hasreached a preset accuracy.

A predetermined time period after administration of the isotopicallylabeled active compound to a subject may be used to provide a highlyaccurate measure of diagnostic value. This period may be determined byusing a sample of control subjects (with and without P. aeruginosainfection) who have been administered isotopically labeled compounds andthen measuring isotopically labeled elements in the exhaled breath ofthe subjects after identified periods of time. The predetermined periodof time is that period during which samples of a subject are taken afteradministration

Following the step of administering the isotopically labeled activecompounds to the subject orally or by pulmonary administration, after anappropriate time period which allows for delivery of the activecompounds to the lung tissue and likely site of infection of P.aeruginosa, if present, the exhaled breath of the subject is analyzed todetect a cleavage product or products, which indicates the presence ofP. aeruginosa in the lungs or other tissue of the subject and whether ornot the P. aeruginosa is predominantly a mucoid or non-mucoid phenotype.The product or products are detected by analyzing a gas sample of theexhaled breath of the subject with a measuring instrument (or a urine,serum or plasma sample for analyzing nitrite, nitrate, NO, cyanate,thiocyanate, biocarbonate levels from cleavage of the active compounds).Such a gas sample can be obtained in a number of ways including, but notlimited to, having the subject exhale or blow into a tube connected tothe measuring instrument. A breath collection bag, a glass vialcontaining a septum or a nasal cannula may be used. The subject breathesdirectly into the breath collection bag or through the septum into theglass vial. In the case of the nasal cannula, such a cannula includes asection of tubing, usually plastic, with two prongs. Each prong isinserted into a nostril and the cannula is then connected to themeasuring instrument. As the subject exhales through the nose, theexhaled air flows through the cannula to the measuring instrument.

The type of measuring instrument used to detect the product or productsdepends upon the type of label, but the present invention may be adaptedrather broadly for virtually any appropriate measuring instrument.Preferably, the instrument is a mass spectrometer gas analyzer, or aninfrared laser spectrometer. For example, if a nitrogen-15 or carbon-13(oxygen-17 or oxygen-18) isotopically-labelled substrate is used, theNitrogen-15 or carbon-13 (oxygen-17 or oxygen-18) isotopically-labelledcleavage product or products can be detected by using a measuringinstrument including, but not limited to a mass spectrometer or a gasanalyzer, which is sensitive to the nitrogen-15 or carbon-13 (oxygen-17or oxygen-18) isotope. In one approach, the ratio of the concentrationof carbon-13 or nitrogen-15 (oxygen-17 or oxygen-18)isotopically-labelled cleavage product or products to the concentrationof carbon-12 or nitrogen-14 (oxygen-16) cleavage product or products isthen determined. Since nitrogen-14 and carbon-12 and oxygen-16 are themore abundant isotopes in nature, nitrogen-14, carbon-12 and oxygen-16atoms are more abundant in unlabelled molecules which are found in theexhaled breath of a patient or subject. Thus, a highercarbon-13/carbon-12, nitrogen-15/nitrogen14, or oxygen 17-18/oxygen-16ratio determined indicates a higher concentration of the carbon-13,Nitrogen-15, oxygen-17 or oxygen 18 isotopically-labelled cleavageproduct or products, which positively indicates the presence of P.aeruginosa in the lungs (or other tissues) of the subject. It is notedthat the ratio (isotopically-labeled atom/non-isotopically labeled atom)obtained after administration of isotopically labeled glycine andmeasurement of the resultant isotopically labeled HCN gas may be usedfurther to determine the existence and extent that P. aeruginosa isphenotypically a mucoid variety.

Preferably, at least one of the cleavage products of glycine is anitrogen-15 isotopically labeled hydrogen cyanide (HCN) gas and urea iscarbon-13 isotopically-labeled carbon dioxide. In the case of nitrates,nitrates and NO precursor compounds, at least one of the cleavageproducts is an isotopically labeled NO gas, N₂O or N₂ gas. In certaininstances, it may be advantageous to label active compounds with morethan one isotopically labeled element such that the cleavage productwould contain more than one isotopically labeled

Examples of measuring instruments which can be used with carbon-13isotopically-labelled carbon dioxide include, but are not limited to, aninfrared spectrometer and an isotope ratio mass spectrometer. Theinfrared spectrometers are well known in the art, and have the advantageof being both rapid and accurate, as well as sensitive. Examples of suchinfrared spectrometers are disclosed in U.S. Pat. No. 5,063,275, whichis incorporated by reference herein.

Alternatively, an analytical assay is described which is based on theuse of Nitrogen-15 labeled expired nitric oxide (NO), N₂O or N₂ gas orC-13 labeled expired CO₂ in the present assay. In this method forexample, isotope ratio mass spectroscopy (EMS) is used as a detectionmethod for N-15, C-13 (also O-17 or O-18) which occurs naturally in theexpired breath of a subject and this ratio is compared with a ratiogenerated for the expired breath of a test subject after administrationof the isotopically labeled active compound (s). A particularlypreferred mass spectromer is a Finnegan Delta Plus XL, which is anisotope ratio mass spectrometer. Non-dispersive infrared spectroscopy(NDIRS) analysis and analysis methods which are well known in the artalso may be employed. HCN gas may be detected/measured using a massspectrometer or laser intrared spectrometer or directly, using achemically integrating dosimeter as described inter alia in U.S. Pat.No. 4,267,023 or after conversion to CO₂ or N₂. Alternatively, HCN, CO₂,CO, NO, N₂O and N₂ may be readily measured using a gas analyzer, such asa Picarro gas analyzer available form Picarro, Inc. Sunnyvale, Calif.

A representative test protocol for the present invention is as follows:isotopically labeled active (nitrate, nitrite, NO, NO precursor, ureaand/or glycine, preferably urea and glycine and optionally one ofnitrate, nitrate, NO precursor) is administered to a subject or patientto be tested. The administration may be by oral or preferably bypulmonary administration, as described in greater detail herein, usingan inhaler or other device adapted to deliver an effective amount(generally, an effective amount within the range of about 0.05 to about25 mg, about 0.25 to about 10 mg, about 0.5 to about 8 mg, about 0.5 toabout 5 mg, about 0.75 to about 3 mg) of isotopically labeled active,preferably isotopically labeled nitrate, nitrite or NO precursor(preferably Nitrogen-15 isotopically labeled nitrite, nitrate, NO orS-nitrosothiol, e.g. S-nitrosoglutathione), urea (preferably carbon-13isotopically labeled urea) and glycine (preferably a Nitrogen-15isotopically labeled glycine) to the lungs of the patient or subject.After an appropriate period of fasting and prior to administration ofthe actives, in certain instances, a number of breaths are taken for thesubject at a predetermined time to produce a “control ratio” or baselineratio of isotopically labeled atom to non-isotopically labeled atom inthe breath of the subject to be tested. Alternatively, a control ratiofor a predetermined time period may be determined using a controlpopulation, rather than the subject of the diagnosis. Just prior to thetaking of breath samples for the test, a dose of isotopically labeledactive (preferably, a combination of actives as described hereinabove)is administered to the subject to be tested either orally or by apulmonary, preferably an intratracheal, route. Breath samples from thesubject are collected after a predetermined time, with the predeterminedtime being significantly different for oral administration relative topulmonary administration of the actives. Ratios of isotopically labeledatoms to non-isotopically labeled atoms are measured/determined from thebreath of the subject to be diagnosed and this ratio is then compared tothe “control ratio”. A measured ratio which is significantly higher thanthe control ratio is evidence of infection by P. aeruginosa, andevidence of increased cleavage of glycine (by measuring cleavageproducts directly or indirectly) indicates that a P. aeruginosainfection is principally phenotypically mucoid. In instances where themeasured ratio of isotopically labeled atom to non-isotopically labeledatom after administration of actives is higher than the control ratio,then P. aeruginosa infectivity is made out. In instances where cleavageof glycine is evidenced, then the presence of a mucoid variety of P.aeruginosa is diagnosed as being present in the patient's lungs.

Advantages of the test are the following: it is practical, sensitive andspecific; the validity of the test is not influenced by stress,exercise, hormone imbalances, or some drugs and medications it is anon-invasive method; it is simple to perform and can be readily used inphysicians' offices or medical laboratories; it is safe since carbon-13,nitrogen-15, and oxygen-17 and -18 are naturally occurring isotopesfound in all carbon-containing and nitrogen-containing substances; itinvolves no radioactivity, and may be used in children and women.

The carbon-13/Nitrogen-15 test is safe, reliable, and specific indiagnosis of P. aeruginosa, measurement of the severity and type(mucoid/non-mucoid) of P. aeruginosa infections in patients. Theinvention is also preferred to monitor P. aeruginosa in CF patients,especially those who are being treated with antibiotics. A preferredembodiment of the invention is a kit containing the necessary materialfor performing the described method. This kit may contain but is notlimited to a source of nitrogen-15, carbon-13, oxygen-17 and/oroxygen-18 isotopically labeled actives, preferably in combination, as anoral or pulmonary dosage form; and a breath collection device. The kitmay also contain a set of patient instructions for its use. In anotherembodiment, the kit may additionally contain a blood collection devicesuch as a lancet or hypodermic needle and vacutainer or a urinecollection vial or other container for the additional determination ofblood (serum or plasma) or urine levels of nitrite and/or nitrate.

Alternatively, at least one of the cleavage products is nitrogen-15isotopically-labeled nitric oxide, N₂O or N₂ (from nitrate, nitrite orNO precursor compound), one is carbon-13 isotopically labeled carbondioxide (from urea), and one is a carbon-13 and/or a nitrogen-15isotopically labeled HCN (or CO₂ or N₂ from HCN). In the case of urea,both carbon-13 isotopically-labelled carbon dioxide and nitrogen-15isotopically-labelled ammonia could be present as cleavage products,providing that the substrate has both labels. Both ammonia and carbondioxide have the advantage of being molecules which are present in theexhaled breath of the subject.

In certain instances, the administered urea may be labeled withcarbon-13, such that the cleavage product is isotopically labeled carbondioxide (CO₂). The isotopically labeled carbon dioxide may be measureddirectly. Ratios of isotopically labeled carbon-13 to non-isotopicallylabeled carbon-12 in the sample breaths may be determined and comparedto control ratios. In other instances, the urea may be labeled withoxygen 17 or 18, such that the cleavage product is isotopically labeledcarbon dioxide (CO₂). The isotopically labeled carbon dioxide may bemeasured directly and ratios of isotopically labeled oxygen-17 or 18 tonon-isotopically labeled oxygen 16 in the sample breaths may bedetermined and compared to a control ratio. The ratio of ¹³CO₂ to ¹²CO2in the CO₂ produced from urease within the bacterium and then exhaledcan be examined directly by mass spectrometry or laser spectroscopy.

Although a number of instruments may be used to measure cleavageproducts in the present invention, certain characteristics of themeasuring device are important. For example, the measuring instrumentused to detect the cleavage product or products should have a number ofcharacteristics. The measuring instrument should be able to measure theconcentration of the product or products extremely rapidly. Furthermore,either the measuring instrument itself, or an associated device, shouldbe able to perform the associated analysis, including providing areadout or in the case where a curve is to be generated, generating thecurve and fitting the curve and providing the analysis of the curve.Such analyses must be performed rapidly. Preferably, the measuringinstrument, alone or in conjunction with the associated device, shouldbe able to measure the concentration and perform the associated analysiswithin about 10 seconds, and most preferably within about 3 seconds,particularly if substantially every exhaled breath of the subject is tobe analyzed.

The term “predetermined time”, “predetermined period” or “predeterminedtime period” (all of which may be used interchangeably within thecontext of their use in describing the present invention) is used todescribe a previously determined (using a control group) period of timeor periods of time at which exhaled breaths (or urine, whole blood,serum or plasma samples) are collected from a subject afteradministration of active compounds in order to analyze for cleavageproducts to determine whether or not the subject has active P.aeruginosa. In the case of active compounds which has been administeredusing pulmonary administration, the predetermined time (period of timecollecting exhaled breaths from a subject) may be a about 15 second toabout 10 minutes, about 30 second to 5 minutes, about 45 seconds to 4minutes, about one minute to 3 minutes, said time period being initiatedany time from about 30 seconds to about an hour, about 1 minute to about30 minutes, about 2 minutes to about 15 minutes about 3 minutes to about10 minutes, about 4 minutes to about 7 minutes after pulmonaryadministration of active compound(s). In the case of oraladministration, the predetermined time period for collecting exhaledbreaths from a subject may be initiated from about 5 minutes to about 5hours after administration, about 10 minutes to about 2 hour, about 15minutes to about 1 hour, about 15 minutes to about 1 hour, about 20minutes to about 45 minutes, about 25 minutes to about 35 minutes afteradministration.

After oral or pulmonary administration, isotopically labeled cleavageproducts such as nitrites, nitrates, NO species, bicarbonate (urea),cyanate and thiocyanate (glycine) from active cleavage may be analyzedfrom urine, serum or plasma samples.

Thus, the predetermined time period refers to the length of a timeperiod at a particular time (generally after an event, especially theadministration of active) required for a cleavage product or products toform and to be exhaled in the breath (or found in the urine, serum orplasma) of the subject. Thus, a number of events must occur. First, theadministered active (s) must be accessible to P. aeruginosa in thelungs. Then, the administered active(s) must be cleaved by the enzymesof P. aeruginosa, if present, to form a cleavage product or products.The cleavage product or products must be exhaled in the breath of thesubject. Finally, the presence of the cleavage product or products mustbe detected in the exhaled breath.

Furthermore, the predetermined time period should be such that enoughbreaths are taken to determine a ratio of isotopically labeled elementsto non-isotopically labeled elements in the exhaled the breaths from thesubject. These may be compared to a control ratio (from the subject or acontrol group, as otherwise described herein) from which a diagnosis ofP. aeruginosa infection or mucoid P. aeruginosa infection is made. Inother embodiments there may be more than one time period such that aseries of measurements are made and form the basis for the curve ofmeasured concentrations. In this aspect which relies on multiplemeasurements (time periods) the concentration will rise rapidlyinitially so that the fitted curve is substantially linear, and willthen plateau after about 10-30 minutes, as the process of formation andexhalation of cleavage product or products reaches a steady state.Eventually, as the administered active is cleaved, the concentration ofcleavage product and isotopically labeled element will decrease. Theanalysis is preferably performed before the curve of measured valuesreaches a plateau.

The term “control ratio” signifies the ratio of isotopically labeledelement to non-isotopically labeled element in a sample obtained fromthe subject prior to administration of active(s) and/or urea or asimilar ratio obtained from a control population, rather than thesubject.

This fitting and analysis of a curve of measured concentrations may bepreferred over other approaches. However, the method of the presentinvention allows repeated breath samples to be rapidly obtained eitherwithin a single time period or multiple time periods and then maximizesboth the speed and the accuracy of analysis by providing a one pointreference number (for the single time period analysis) above whichdiagnosis of active infection may be made or in the case of multipletime periods, fitting the measured values to a curve and thencalculating the rate of increase of the curve, which evidences theinfection and its intensity.

Any method for identifying the concentration of isotopically labelednitric oxide, N₂O or N₂ (from nitrate, nitrate, NO denitrification),carbon dioxide and/or ammonia (from cleavage of urea) gas or HCN gas(from cyanide synthase) can be used to determine the existence (orabsence) and phenotype (mucoid or non-mucoid) of P. aeruginosa in thelungs (or other tissues) of a patient or subject. The measurement ofisotopically labeled gas as a cleavage product by action of P.aeruginosa on the actives is evidence of the existence (or absence) ofP. aeruginosa in the lungs (and other tissues) of the subject or theexistence (or absence) of a mucoid/non-mucoid strain of M. tuberculosisin the lungs (or other tissues) of the subject. Thus, where bothisotopically labeled glycine and urea or other active are administeredto a subject to be diagnosed, evidence of cleavage of none of theactives is strong evidence of the subject being P. aeruginosa free,evidence of cleavage of urea or other actives but not glycine indicatesthe existence of a P. aeruginosa infection which is non-mucoid.

In the present invention it is preferred to determine a ratio of anisotopically labeled element (carbon, nitrogen, oxygen) to anon-isotopically labeled element in a cleavage product (gas) beinganalyzed. For example, if nitric oxide (NO), N₂O or N₂ is being measuredas a cleavage product pursuant to administration of nitrate, nitrite orNO precursor, a ratio of nitrogen-15 to nitrogen-14 in the breath of asubject is determined. This may be determined readily using massspectroscopy or infrared laser spectroscopy. In preferred aspects of theinvention, a ratio of Nitrogen-15 to Nitrogen-14 in gasses exhaled by asubject to be diagnosed before administration of actives is determinedas a baseline ratio. This ratio is generally below 1%

Specifically, an exemplary method of analysis involves the followingsteps. A plurality of samples of exhaled breath of the subject iscollected rapidly, on the order of one sample about every few seconds orso, preferably such that at least a majority, and most preferablysubstantially all of the exhaled breaths of the subject at apredetermined time for a predetermined period(s) are sampled. Next, theconcentration of a cleavage product is measured and the concentration ofan isotopically labeled element, such as nitrogen-15, carbon-13,oxygen-17 or oxygen-17 is compared with its naturally occurringcounterpart (e.g. respectively, nitrogen-14, carbon-12 and oxygen-16) inthe breath of the subject. Where the ratio of isotopically-labeledelement to naturally occurring element is approximately 0 orapproximately a control ratio (the control ratio is based uponmeasurements taken in the subject prior to administration of actives),then P. aeruginosa is not present. In cases where the ratio ofisotopically-labeled element to naturally occurring element is above apredetermined value (e.g. established from control groups) measurementsabove the predetermined value and/or increases of the ratio as afunction of time, evidences the existence of P. aeruginosa. Where theratio of isotopically-labeled element or the direct measurement of HCNevidences the cleavage of glycine by cyanide synthase, then a mucoid P.aeruginosa infection is diagnosed.

Although measuring and analyzing exhaled breaths from a subject for asingle predetermined period represents a preferred approach todetermining the existence or absence of a P. aeruginosa infection,alternative approaches also may be used. In instances where a number ofmeasurements of exhaled breath from the subject are taken from differentperiods, a curve may be fitted or generated from the measuredconcentrations. A curve may be fitted to the measured concentrations. Ifthe ratio remains flat at the x-axis (essentially 0 or close to 0-basedupon the subject or a control group) as a function of time, the presenceof P. aeruginosa is ruled out. The rate of rise of the curve may bedetermined by calculating the integral or by derivation (calculation ofthe derivative), preferably after the measurement of the concentrationof cleavage product(s) in each sample. The analysis of the curveindicates the level of P. aeruginosa activity in the lungs of thesubject. A rapid rise in the measured concentrations (a steeper curve),would evidence a high level of P. aeruginosa activity in the subject,whereas a slower rise in the measured concentrations (a shallower curve)would evidence a lower level of P. aeruginosa activity. If the P.aeruginosa infection is systemic, a greater period of time will berequired for absorption and distribution of the labeled compound to thesite of infection and release of labeled gasses. The technique will alsoprove useful in monitoring the responses of the P. aeruginosa infectionto drugs, if the drugs are effective then the bacterial load (measuredas either the rate of labeled gas increase, or the value of the plateau,will continue to decrease—if the drugs are ineffective due toresistance, then this will not happen and so alternative drug therapieswill be tried.

In an analogous manner, if isotopically labeled actives (preferablyhaving different elements labeled so that cleavage products of oneactive will be distinguishable from cleavage products of another active)are used, these can provide evidence of the existence (or absence) of P.aeruginosa and the type of infection (mucoid vs. non-mucoid) whichpredominates.

The single point (predetermined time period) approach to diagnosticanalysis has a number of advantages, the major ones being the ease ofuse and rapid nature of the diagnosis. This approach also provides adiagnostic method which can be used in a clinic or even a doctor'soffice. A single calculation may be made by taking a number of exhaledbreaths from the patient or subject for the predetermined period andthen analyzing for isotope-labeled elements in the sample, providing aration of isotopically-labeled elements to non-isotopically-labeledelements and comparing that ratio to a predetermined ratio obtained fromthe subject or from a control group.

In other approaches, the calculation of a derivative from a graphproduced from a number of collection samples (from varying time periods)which provides a number of data points has advantages over other methodsof analysis, such as the calculation of an integral. First, thecalculation of the derivative does not require a reference breath sampleto be obtained before active is administered to the subject. Since thederivative represents the rate of increase of the measuredconcentrations of a cleavage product or products, the startingconcentration of that cleavage product or products is unimportant.However, the initial concentration of the cleavage product or productsin the reference breath sample is important for the proper calculationof the integral, since such an initial concentration represents abackground value which must be subtracted from the measuredconcentrations after administration of the active.

After the resultant measurement has reached a predetermined level ofaccuracy, or after a predetermined time period has elapsed, no moresamples are collected.

The present method utilizing a breath assay has a number of advantages.First, the exhaled breath of the subject can be analyzed in real time;that is, there is relatively little delay between the time the P.aeruginosa activity takes place, and the time such activity is measured.Second, the samples of exhaled breath are obtained rapidly and areanalyzed immediately in a manner which substantially increases theaccuracy of the results. Depending on method, one or multiple samplesmay be obtained. In general, a single sample (from a number of exhaledbreaths) represents a convenient method which exhibits ease of use andpatient compliance. In contrast, obtaining multiple samples from thesubject increases the accuracy of the test. There is also lessstatistical error since many samples are collected. In addition, in thisaspect, since samples are preferably collected until a preset level ofaccuracy is reached, ambiguous results can be substantially eliminated,preventing the need for repeating the test.

The readout of isotopic ratios can be performed by sensitive gas massspectrometry analysis, but also laser spectroscopy techniques which mayallow for more compact and portable devices. In certain aspects of theinvention, especially where a ratio of isotopically labeled element tonon-isotopically labeled element in a gas is to be used in the analysis,a Finnegan Delta Plus XL™ Mass Spectromer may be used. Collection ofexhaled gases (which include gaseous cleavage products) from the subjectwhen the cleaved products are gases may be effected using a standard gascollection bag, using a glass vial with a septum (the subject simplyblows into the vial through the septum, or using any other method forcollecting breaths from the subject. It is also noted that isotopicallylabeled Nitrogen-15 present in nitrites, NO, nitrates, cyanates andthiocyates in the urine, serum or plasma of the subject fromcleavage/reduction of isotopically labeled nitrates, nitrites, NOprecursor compounds (denitrification pathway), or glycine (cyanidesynthase) may also be analyzed according to the present invention. Aratio of isotopically labeled Nitrogen-15 to non-isotopically labeledNitrogen in the urine, whole blood, serum or plasma sample after theadministration of the isotopically labeled active compound may bedetermined and compared to control levels taken from urine, whole blood,serum or plasma of the subject prior to labeled active administration.Alternatively, a control group can be used to establish control levelsof isotopically labeled nitrites, nitrates, NO, cyanates andthiocyanates in urine, serum or plasma levels and appropriate ratios forcomparison purposes. In like fashion, isotopically labeled (carbon-13)CO₂ as dissolved carbonate/bicarbonate in the urine, whole blood, serumor plasma also may be analyzed from the cleavage of isotopically labeled(carbon-13) urea (urease) by P. aeruginosa.

In the present method, isotopically labeled active compounds may beadministered by oral or preferably, by a pulmonary (e.g. intratracheal)route of administration. In the case of oral administration, theisotopically labeled actives, either alone or in combination areadministered orally to a subject to be tested for evidence of P.aeruginosa infection. The isotopically labeled active compounds may beadministered in standard oral dosage form, preferably as an immediaterelease dosage form or as an enteric dosage form (especially whenadministered in combination with urea), in combination with apharmaceutically acceptable carrier, additive or excipient. Oralformulations which include urea may be formulated in enteric dosage formto promote release in the small intestine (duodenum, jejunum, ileum) orin combination with a urease inhibitor to inhibit the action of H.pyrlori urease on administered urea prior to its such as acetohydroxamicacid (Lithostat), a bismuth salt such as bismuth nitrate, bismuthcarbonate, bismuth salicylate or bismuth citrate, a proton pumpinhibitor such omeprazole (Prilosec), esomeprazole (Nexium),lansoprazole (Prevacid), pantoprazole (Protonix) and rabeprazole sodium(Aciphex), or a natural product extract from ranunculus repens, to avoidany action by urease from H. pylori in the gastrointestinal tract, morespecifically, the stomach.

Thus, the present invention also relates to pharmaceutical compositionsin oral dosage form comprising effective amounts of isotopically labeledglycine, urea, at least one compound selected from the group consistingof a nitrate salt, a nitrite salt, a NO precursor (e.g. S-nitrosothiolcompound such as an alkylthionitrile, S-glutathione,S-nitroso-N-acetyl-penicillamine) or their pharmaceutically acceptablesalts thereof and a urease inhibitor (not isotopically labeled),optionally in combination with a pharmaceutically acceptable carrier,additive or excipient. Compositions for oral administration includepowders or granules, suspensions or solutions in water or non-aqueousmedia, sachets, capsules or tablets. Thickeners, diluents, flavorings,dispersing aids, emulsifiers or binders may be desirable.

In preferred aspects of the invention, the isotopically labeled activecompounds are administered to the lungs of the subject via pulmonaryadministration, including intratracheal administration. Thepharmaceutical composition of the invention for pulmonary administrationis usually used as an inhalant. The composition can be formed into drypowder inhalants, inhalant suspensions, inhalant solutions, encapsulatedinhalants and like known forms of inhalants. Such forms of inhalants canbe prepared by filling the pharmaceutical composition of the inventioninto an appropriate inhaler such as a metered-dose inhaler, dry powderinhaler, atomizer bottle, nebulizer etc. before use. Of the above formsof inhalants, powder inhalants may be preferable.

When the pharmaceutical composition of the invention is used in the formof a powder, the mean particle diameter of the powder is not especiallylimited but, in view of the residence of the particles in the lungs, ispreferably that the particles fall within the range of about 0.1 to 20μm, and particularly about 1 to 5 μm. Although the particle sizedistribution of the powder pharmaceutical composition of the inventionis not particularly limited, it is preferable that particles having asize of about 25 μm or more account for not more than about 5% of theparticles, and preferably, 1% or less to maximize delivery into thelungs of the subject.

The pharmaceutical composition in the form of a powder of the inventioncan be produced by, for example, using the drying-micronization method,the spray drying method and standard pharmaceutical methodology wellknown in the art.

By way of example without limitation, according to thedrying-pulverization method, the pharmaceutical composition in the formof a powder can be prepared by drying an aqueous solution (or aqueousdispersion) containing the isotopically labeled active compounds ormixtures thereof and excipients which provide for immediate release inpulmonary tissue and microparticulating the dried product. Stated morespecifically, after dissolving (or dispersing) a pharmaceuticallyacceptable carrier, additive or excipient in an aqueous medium, glycine,urea, or mixtures of glycine and urea, and optionally at least onecompound selected from the group consisting of a nitrate salt, a nitritesalt or a NO precursor compound (S-nitrosothiol compound, e.g., analkylthionitrile, S-glutathione, S-nitroso-N-acetylpenicillamine), ortheir pharmaceutically acceptable salts, all isotopically labeled, ineffective amounts are added and dissolved (or dispersed) by stirringusing a homogenizer, etc. to give an aqueous solution (or aqueousdispersion). The aqueous medium may be water alone or a mixture of waterand a lower alcohol. Examples of usable lower alcohols include methanol,ethanol, 1-propanol, 2-propanol and like water-miscible alcohols.Ethanol is particularly preferable. After the obtained aqueous solution(or aqueous dispersion) is dried by blower, lyophilization, etc., theresulting product is pulverized or microparticulated into fine particlesusing jet mills, ball mills or like devices to give a powder having theabove mean particle diameter. If necessary, additives as mentioned abovemay be added in any of the above steps.

According to the spray-drying method, the pharmaceutical composition inthe form of a powder of the invention can be prepared, for example, byspray-drying an aqueous solution (or aqueous dispersion) containingglycine, urea or mixtures thereof and optionally at least one compoundselected from the group consisting of a nitrate salt, a nitrite salt ora NO precursor compound (S-nitrosothiol compound, e.g., analkylthionitrile, S-glutathione, S-nitroso-N-acetylpenicillamine) andexcipients, additives or carriers for microparticulation. The aqueoussolution (or aqueous dispersion) can be prepared following the procedureof the above drying-micronization method. The spray-drying process canbe performed using a known method, thereby giving a powderypharmaceutical composition in the form of globular particles with theabove-mentioned mean particle diameter.

The inhalant suspensions, inhalant solutions, encapsulated inhalants,etc. can also be prepared using the pharmaceutical composition in theform of a powder produced by the drying-micronization method, thespray-drying method and the like, or by using a carrier, additive orexcipient and isotopically labeled active(s) that can be administeredvia the lungs, according to known preparation methods.

Furthermore, the inhalant comprising the pharmaceutical composition ofthe invention is preferably used as an aerosol. The aerosol can beprepared, for example, by filling the pharmaceutical composition of theinvention and a propellant into an aerosol container. If necessary,dispersants, solvents and the like may be added. The aerosols may beprepared as 2-phase systems, 3-phase systems and diaphragm systems(double containers). The aerosol can be used in any form of a powder,suspension, solution or the like.

Examples of usable propellants include liquefied gas propellants,compressed gases and the like. Usable liquefied gas propellants include,for example, fluorinated hydrocarbons (e.g., CFC substitutes such asHCFC-22, HCFC-123, HFC-134a, HFC-227 and the like), liquefied petroleum,dimethyl ether and the like. Usable compressed gases include, forexample, soluble gases (e.g., carbon dioxide, nitric oxide), insolublegases (e.g., Nitrogen) and the like.

The dispersant and solvent may be suitably selected from the additivesmentioned above. The aerosol can be prepared, for example, by a known2-step method comprising the step of preparing the composition of theinvention and the step of filling and sealing the composition andpropellant into the aerosol container.

As a preferred embodiment of the aerosol according to the invention, thefollowing aerosol can be mentioned: Examples of the compounds to be usedinclude isotopically labeled isoniazid, isotopically labeled urea ormixtures thereof. As propellants, fluorinated hydrocarbons such asHFC-134a, HFC-227 and like CFC substitutes are preferable. Examples ofusable solvents include water, ethanol, 2-propanol and the like. Waterand ethanol are particularly preferable. In particular, a weight ratioof water to ethanol in the range of about 0:1 to 10:1 may be used.

The aerosol of the invention contains excipient in an amount rangingfrom about 0.01 to about 10⁴ wt. % (preferably about 0.1 to 10³ wt. %),propellant in an amount of about 10² to 10⁷ wt. % (preferably about 10³to 10⁶ wt. %), solvent in an amount of about 0 to 10⁶ wt. % (preferablyabout 10 to 10⁵ wt. %), and dispersant in an amount of 0 to 10³ wt. %(preferably about 0.01 to 10² wt. %), relative to the weight ofisoniazid and/or urea which is included in the final composition.

The pharmaceutical compositions of the invention are safe and effectivefor use in the diagnostic methods according to the present invention.Although the dosage of the composition of the invention may varydepending on the type of active substance administered (isoniazid, ureaor mixtures thereof) as well as the nature (size, weight, etc.) of thesubject to be diagnosed, the composition is administered in an amounteffective for allowing the pharmacologically active substance to becleaved to cleavage products to be measured. For example, thecomposition is preferably administered such that the active ingredientcan be given to a human adult in a dose of about 0.001 to about 100 mg,about 0.01 mg to about 25 mg, about 0.05 mg to about 15 mg, about 0.1 mgto about 10 mg, about 0.5 mg to about 5 mg, about 1 to about 3 mg, andgiven in a single dose

The form of the pharmaceutical composition of the invention such as apowder, solution, suspension etc. may be suitably selected according tothe type of substance to be administered and the action of a targetenzyme on the isotopically labeled active compound(s).

As an administration route, direct inhalation via the mouth using aninhaler is usually preferable. Since the pharmaceutical composition ofthe invention allows direct local administration into the airways and inparticular, directly to pulmonary tissue, the active substance containedtherein produces immediate effects. Furthermore, the composition isformulated as an immediate release product so that cleavage and analysiscan begin soon after administration.

ADVANTAGES

The present invention allows diagnosis of a P. aeruginosa infection in aCF patient or subject, including a mucoid P. aeruginosa infection in asimple diagnostic test, including a breath test. The diagnosis is rapidand effective and displays a number of ancillary advantages as well.

The present invention also always the monitoring of a P. aeruginosainfection in the lungs of a CF patient, during therapy to determine theimpact that an antibiotic regimen is having on resolving the infection.

The present invention represents a rapid, one-stop, easily administeredtest. The present approach of obtaining a sample from the lungs of theCF patient and processing the sample to determine the existence of a P.aeruginosa infection is laborious, time consuming and not costeffective. The present approach creates logistical issues. The presentdiagnostic test would be administered ‘all in one go’ obviating the needfor precise recall and would reduce/remove laboratory error/chain ofcustody concerns. The present system allow faster treatment of infectedand potentially infectious patients. Finally, administration of a tracertablet and collecting breath or urine afterwards needs lower levelmedical skills, making widespread testing by paramedics possible.Samples can easily be mailed to central facilities.

Allows Rapid Detection of Mucoid formation of P. aeruginosa. This ispotentially quite harmful to the CF patient and the present method notonly can identify mucoid P. aeruginosa infections with great accuracy,but can monitor the progression of a non-mucoid P. aeruginosa infectionso that steps can be taken to modify a therapeutic regiment for a moresuccessful outcome.

The following examples provide insight into the use of the presentinvention. The examples are simply that, exemplary, and are not to beconstrued to limit the present invention in any way.

EXAMPLES Administration of Diagnostic Test for Presence of P. aeruginosaInfection

There are a Number of Potential Embodiments of the Diagnostic Assay

Example 1 Inhaled Administration of Labeled Tracer

A. A first embodiment is to administer ¹³C-urea by inhalation (such as adry powder inhaler as described above) in an amount of 0.1 to 10 mg, andthe ratio of ¹³CO₂ to ¹²CO₂ in exhaled breath sample 1 to 60 minutesafterwards (to allow for conversion) is determined by mass spectrometryor laser spectroscopy. The ratio of ¹³CO₂ to ¹²CO₂ is compared to thatof a breath sample obtained before compound inhalation, and an increasein this ratio is indicative of P. aeruginosa infection.

B. A second embodiment relates to replace the use of urea with the useof ¹⁵N-glycine as in the example A above, with analysis of the amount ofHCN (labeled with ¹⁵N) compared to HCN in a sample prior to glycineadministration in an exhaled breath sample 1 to 60 minutes afteradministration is determined by mass spectrometry or laser spectroscopy.The ratio of ¹⁵N to ¹⁴N is compared to that of a breath sample obtainedbefore glycine inhalation, and an increase in this ratio is indicativeof P. aeruginosa infection which is predominantly mucoid.

C. Another embodiment is to administer both ¹³C-urea and ¹⁵N-glycine andindependently analyse the ratio of ¹³CO2 to ¹²CO₂ and of ¹⁵N to ¹⁴N inexhaled breath sample 1 to 60 minutes after is determined by massspectrometry or laser spectroscopy. The ratio of ¹³CO₂ to ¹²CO₂ (fromurea) and ¹⁵N to ¹⁴N (from glycine) is compared to that of a breathsample obtained before compound inhalation, and so determination ofmycobacterial disease and predominance of mucoid phenotype can besimultaneously determined.

D. If desired, isotopically labeled ¹⁵N-nitrate, nitrite salts or NOprecursor compounds may be administered to the CF patient, optionally incombination with isotopically labeled urea and/or glycine and theresultant analysis of isotopically labeled metabolites performed.Elevated levels of isotopically labeled metabolites may evidence thepresence of a P. aeruginosa infection, and in particular, an infectionhaving a mucoid phenotype.

E. If desired, as described hereinabove, other active compounds andanalytes may be measured, such as isotopically labeled N₂O, NO or N₂gases, or nitrites, nitrates, NO, bicarbonate/carbonate, cyanate,thiocynate in a blood sample (serum, plasma), or a urine sample.

Example 2 Oral Administration of Labeled Tracer

1) Oral Administration of Labeled Tracer

A.) One approach is to administer ¹³C-urea by in an oral dosage in anamount of about 0.1 to 100 mg, and the ratio of ¹³CO₂ to ¹²CO₂ inexhaled breath sample 1 to 180 minutes (also within the range of about15-30 minutes to 60 minutes) after administration is determined by massspectrometry or laser spectroscopy. The ratio of ¹³CO₂ to ¹²CO₂ iscompared to that of a breath sample obtained before compoundadministration, and an increase in this ratio is indicative of P.aeruginosa infection. It may be desirable to preclude the potential forH. pylori interference with this assay by either enterically coating theurea capsule/tablet so that labeled urea does not come into contact withH. pylori and/or administration of known inhibitors of H. pylori ureasesuch as bismuth salts, as otherwise described herein.

B) Another approach is to replace use of urea with the use of¹⁵N-glycine with analysis of the ratio of ¹⁵N to ¹⁴N in exhaled breathsample 1 to 200 minutes after is determined by mass spectrometry orlaser spectroscopy and compared to a predetermined ratio of ¹⁵N to ¹⁴Nin a breath sample obtained from the patient or subject prior toadministration of glycine. In addition, the amount of isotopicallylabeled HCN may be measured directly and compared with HCN in a breathsample obtained before compound administration. An increase in thisratio in both instances is indicative of P. aeruginosa disease that islikely to have a mucoid phenotype.

C) Another is to orally administer both ¹³C-urea and ¹⁵N-glycine andindependently analyse the ratio of ¹³CO₂ to ¹²CO₂ (from urea) and of ¹⁵Nto ¹⁴N (in HCN from glycine) in exhaled breath sample 1 to 120 minutesafter is determined by mass spectrometry or laser spectroscopy. A ureasinhibitor is preferably included to limit the possibility of urease inthe GI tract of the patient metabolizing the administered urea before itgets to the lungs. Note that isotopically labeled HCN may be measureddirectly and compared with HCN in the breath of the CF patient prior toglycine administration. The ratio of ¹³CO2 to ¹²CO2 (from urea) and ¹⁵Nto ¹⁴N (from glycine) is compared to that of a breath sample obtainedbefore compound administration, and so determination of P. aeruginosadisease and likely predominance of mucoid phenotype are present.

D. If desired, isotopically labeled (nitrogen-15) nitrate, nitrite saltsor NO precursor compounds may be administered to the CF patient orally,optionally in combination with isotopically labeled urea (preferablyincluding a urease inhibitor as otherwise described herein) and/orglycine and the resultant analysis of isotopically labeled metabolitesperformed. Elevated levels of isotopically labeled metabolites mayevidence the presence of a P. aeruginosa infection, and in particular,an infection having a mucoid phenotype.

E. If desired, as described hereinabove, other active compounds may beorally administered and analytes may be measured, such as isotopicallylabeled N₂O, NO or N₂ gases, or nitrites, nitrates, NO,bicarbonate/carbonate, cyanate, thiocynate in a blood sample (serum,plasma), or a urine sample, and analysis performed.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

REFERENCES

-   1. Deturk, W. E. The adaptive formation of urease by washed    suspensions of Pseudomonas aeruginosa. J Bacteriol 70, 187-91    (1955).-   2. Jahns, T. Regulation of urea uptake in Pseudomonas aeruginosa.    Antonie Van Leeuwenhoek 62, 173-9 (1992).-   3. Karmali, K., Karmali, A., Teixeira, A. & Curto, M. J. The use of    Fourier transform infrared spectroscopy to assay for urease from    Pseudomonas aeruginosa and Canavalia ensiformis. Anal Biochem 331,    115-21 (2004).-   4. Sundstrom, J., Jacobson, K., Munck-Wikland, E. & Ringertz, S.    Pseudomonas aeruginosa in otitis exteina. A particular variety of    the bacteria? Arch Otolaryngol Head Neck Surg 122, 833-6 (1996).-   5. Deretic, V. in Persistent Bacterial Infections (ed. Nataro, J.    P., Blaser, M. J., Cunningham-Runddles, S.) 305-326 (ASM Press,    Washington, D.C., 2000).-   6. Goldberg, J. B. & Pier, G. B. The role of the CFTR In    susceptibility to Pseudomonas aeruginosa infections in cystic    fibrosis. Trends in Microbiology 8, 514-20 (2000).-   7. FitzSimmons, S. C. The changing epidemiology of cystic fibrosis.    Journal of Pediatrics 122, 1-9 (1993).-   8. Govan, J. R. W. & Deretic, V. Microbial pathogenesis in cystic    fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.    Microbiological Reviews 60, 539-574 (1996).-   9. Martin, D. W. et al. Mechanism of conversion to mucoidy in    Pseudomonas aeruginosa infecting cystic fibrosis patients.    Proceedings of the National Academy of Sciences of the United States    of America 90, 8377-8381 (1993).-   10. Fyfe, J. A. & Govan, J. R. Alginate synthesis in mucoid    Pseudomonas aeruginosa: a chromosomal locus involved in control.    Journal of General Microbiology 119, 443-50 (1980).-   11. McAvoy, M. J. et al. Isolation of mucoid strains of Pseudomonas    aeruginosa from non-cystic-fibrosis patients and characterisation of    the structure of their secreted alginate. Journal of Medical    Microbiology 28, 183-9 (1989).-   12. Ramsey, D. M. & Wozr˜iaU, D. J. ^(T)Jndersta.˜ding the control    of Pseudomonas aeruginosa alginate synthesis and the prospects for    management of chronic infections in cystic fibrosis. Molecular    Microbiology 56, 309-22 (2005).-   13. Pedersen, S. 5., Hoiby, N., Espersen, F. & Koch, C. Role of    alginate in infection with mucoid Pseudomonas aeruginosa in cystic    fibrosis. Thorax 47, 6-13 (1992).-   14. Koch, C. & Hoiby, N. Pathogenesis of cystic fibrosis. Lancet    341, 1065-9 (1993).-   15. Pedersen, S. S. Lung infection with alginate-producing, mucoid    Pseudomonas aeruginosa in cystic fibrosis. APMIS 100(Suppl. 28),    1-79 (1992).-   16. Firoved, A. M., Ornatowski, W. & Deretic, V. Microarray analysis    reveals induction of lipoprotein genes in mucoid Pseudomonas    aeruginosa: implications for inflammation in cystic fibrosis.    Infection & Immunity 72, 5012-8 (2004).-   17. Koch, C. Early infection and progression of cystic fibrosis lung    disease. Pediatric Pulmonology 34, 232-6 (2002).-   18. Bonfield, T. L. et al. Inflammatory cytokines in cystic fibrosis    lungs. American Journal of Respiratory & Critical Care Medicine 152,    2111-2118 (1995).-   19. Cobb, L. M., Mychaleckyj, J. C., Wozniak, D. J. &    Lopez-Boado, Y. S. Pseudomonas aeruginosa flagellin and alginate    elicit very distinct gene expression patterns in airway epithelial    cells: implications for cystic fibrosis disease. J Immunol 173,    5659-70 (2004).-   20. Castric, P. A. Glycine metabolism by Pseudomonas aeruginosa:    hydrogen cyanide biosynthesis. J Bacteriol 130, 826-31 (1977).-   21. Castric, P. A. Hydrogen cyanide production by Pseudomonas    aeruginosa at reduced oxygen levels. Can J Microbiol 29, 1344-9    (1983).-   22. Firoved, A. M. & Deretic, V. Microarray analysis of global gene    expression in mucoid Pseudomonas aeruginosa. J Bacteriol 185,    1071-81 (2003).-   23. Gallagher, L. A. & Manoil, C. Pseudomonas aeruginosa PAO1 kills    Caenorhabditis elegans by cyanide poisoning. J Bacteriol 183,    6207-14 (2001).-   24. Carterson, A. J. et al. The transcriptional regulator AlgR    controls cyanide production in Pseudomonas aeruginosa. J Bacteriol    186, 6837-44 (2004).

The invention claimed is:
 1. A method for diagnosing a presence ofPseudomonas aeruginosa in the lungs of a subject, including the stepsof: (a) administering an effective amount of at least one isotopicallylabeled active agent selected from the group consisting of a nitratesalt, a nitrite salt, nitric oxide (NO), a nitric oxide precursorcompound, urea and glycine or their pharmaceutically acceptable salts tothe subject, said active agent being acted upon by P. aeruginosa to forman isotopically labeled cleavage product or metabolite; and (b)collecting breath, urine, whole blood, plasma or serum samples from thesubject; and (c) analyzing said samples to determine a concentration orconcentrations of said cleavage product(s) or metabolite(s), saidconcentration(s) indicating the presence or absence of P. aeruginosaincluding optionally, a presence or absence of mucoid phenotype P.aeruginosa, in the lungs of said patient.
 2. The method according toclaim 1 wherein said active agent is selected from the group consistingof sodium nitrate, potassium nitrate, sodium nitrite, potassium nitrite,nitric oxide, a pharmaceutically acceptable S-nitrosothiol compound,urea, glycine, pharmaceutically acceptable salts thereof and mixturesthereof.
 3. The method according to claim 2 wherein said S-nitrosothiolcompound is an alkylthionitrile.
 4. The method according to claim 1wherein said nitric oxide precursor compound is S-glutathione orS-nitroso-N-acetylpenicillamine.
 5. The method according to claim 1wherein said isotopically labeled active agent is a mixture of urea,glycine or a pharmaceutically acceptable salt thereof, and optionally,at least one compound selected from the group consisting of anisotopically labeled nitrate salt, nitrite salt, nitric oxide andS-nitrosothiol compound.
 6. The method according to claim 1 wherein saidsample is the breath of said subject taken for a predetermined time. 7.The method according to claim 1 wherein said active agent isisotopically labeled with carbon-13, nitrogen-15, oxygen-17, oxygen-18or mixtures thereof.
 8. The method according to claim 7 wherein saidactive agent is urea isotopically labeled with carbon-13, nitrogen-15,oxygen-17 or oxygen-18.
 9. The method according to claim 8 wherein saidurea is isotopically labeled with carbon-13 or nitrogen-15.
 10. Themethod according to claim 1 wherein said active agent is glycineisotopically labeled with nitrogen-15 or carbon-13.
 11. The methodaccording to claim 10 wherein said glycine is isotopically labeled withnitrogen-15.
 12. The method according to claim 1 wherein said urea isisotopically labeled with carbon-13 and said glycine is isotopicallylabeled with nitrogen-15.
 13. The method according to claim 1 whereinsaid cleavage product or metabolite is a gas.
 14. The method accordingto claim 13 wherein said gas is N₂, N₂O, NO, CO₂, HCN or mixturesthereof.
 15. The method according to claim 14 wherein said gas is amixture of isotopically labeled CO₂, from the action of P. aeruginosa onurea, and HCN, from the action of P. aeruginosa on glycine andoptionally, at least one gas selected from the group consisting of N₂,N₂O and NO.
 16. The method according to claim 13 wherein a measuredconcentration of said gas is indicative of a P. aeruginosa infection inthe lungs of said patient.
 17. The method according to claim 14 whereinsaid gas is HCN or includes HCN and a measured concentration of said HCNgas at a radiometric ratio of at least 1.25 is indicative of a mucoidphenotype of P. aeruginosa in the lungs of said patient.
 18. The methodaccording to claim 1 wherein said isotopically labeled cleavage productor metabolite is selected from the group consisting of nitrate, nitrite,dissolved nitric oxide, carbonate/bicarbonate, cyanate, thiocyanate andmixtures thereof, which is measured in the urine, whole blood, serum orplasma of the patient.
 19. The method according to claim 18 wherein saidcleavage product or metabolite is cyanate, thiocyanate or mixturesthereof or includes cyanate, thiocyanate or mixtures thereof and ameasured concentration of said cleavage product or metabolite in aradiometric ratio of at least about 1.25 is indicative of a mucoidphenotype of P. aeruginosa.
 20. The method according to any of claims1-16 wherein said analyzing step comprises comparing at least one ratioof isotopically labeled element(s) to non-isotopically labeledelement(s) in said exhaled breath of said subject to a control ratio ofisotopically labeled element(s) to non-isotopically labeled elements inthe exhaled breath of said subject or a control group prior toadministration of said isotopically labeled active agent.
 21. The methodaccording to claim 1 wherein said isotopically labeled active agent(s)is delivered to the lungs by pulmonary route of administration.
 22. Themethod according to claim 1 wherein said isotopically labeled activeagent(s) is delivered by oral route of administration.
 23. The methodaccording to claim 22 wherein said active agent is or includes urea andsaid active agent is administered by oral route in combination with aurease inhibitor.
 24. The method according to claim 22 or wherein saidactive agent is urea or includes urea and is administered by oral routein an enteric capsule.
 25. The method according to claim 1 whereinisotopically labeled glycine and urea are both administered to saidsubject, alone or in combination with at least one additionalisotopically labeled active agent selected from the group consisting ofa nitrate salt, a nitrite salt, NO gas and a NO precursor compound, andwherein the absence of glycine cleavage product, urea cleavage and othercleavage products is evidence of the absence of a P. aeruginosainfection in said subject.
 26. The method according to claim 1 whereinisotopically labeled glycine and urea are both administered to saidsubject, alone or in combination with at least one additionalisotopically labeled active agent selected from the group consisting ofa nitrate salt, a nitrite salt, NO gas and a NO precursor compound, andwherein the absence of glycine cleavage product and the presence of ureacleavage product and optionally, other cleavage products is evidence ofa non-mucoid P. aeruginosa infection in said subject.
 27. The methodaccording to claim 1 wherein isotopically labeled glycine and urea areboth administered to said subject, alone or in combination with at leastone additional isotopically labeled active agent selected from the groupconsisting of a nitrate salt, a nitrite salt, NO gas and a NO precursorcompound, and wherein the presence of glycine cleavage product and thepresence of urea cleavage product and optionally, other cleavageproducts is evidence of a mucoid P. aeruginosa infection in saidsubject.
 28. The method according to claim 1 wherein said urea iscleaved to produce carbon dioxide or ammonia.
 29. The method accordingto claim 1 comprising optional steps of fitting the concentrations ofsaid cleavage products obtained to a curve; and analyzing the curve or aplateau of said curve to determine the extent of infection.
 30. A methodfor diagnosing a urease-positive bacterial infection in the lungs of asubject comprising: administrating to the lungs of the subject, aneffective amount isotopically labeled urea; collecting one or moresamples of exhaled breath from the subject; and analyzing said samplesto determine a concentration of isotopically labeled CO₂ in saidsamples; said concentration indicating the presence or absence of theurease-positive bacterial infection in the lungs of the subject.
 31. Themethod of claim 30, wherein the isotopically labeled urea is ¹³C-urea.32. The method of claim 30, wherein said samples are taken for apredetermined time.
 33. The method of claim 30, wherein said gas is¹³CO₂.
 34. The method of claim 31, wherein the analysis comprisescomparing the ratio of ¹³CO₂ to ¹²CO₂ in said samples to the ratio of¹³CO₂ to ¹²CO₂ in a sample obtained from the subject prior to theadministration of the isotopically labeled urea.
 35. The method of claim31, wherein an increase in the ratio of ¹³CO₂ to ¹²CO₂ in said samplesto the ratio of ¹³CO₂ to ¹²CO₂ in a sample obtained from the subjectprior to the administration of the isotopically labeled urea.